Protein kinases are involved in signal transduction pathways linking growth factors, hormones and other cell regulation molecules to cell growth, survival and metabolism under both normal and pathological conditions. The superfamily of protein kinases includes protein kinase A and protein kinase C, as well as the more recently discovered protein kinase B (PKB).
PKB is a newly recognized anti-apoptotic protein kinase whose activity is strongly elevated in human malignancies. PKB was originally discovered as a viral oncogene v-Akt in rat T-cell leukemia. It was later established that v-Akt is the oncogenic version of a cellular enzyme PKB/c-Akt, in which a truncated viral group specific antigen, gag, is fused in frame to the full length Akt-1 and is membrane bound whereas PKB/c-Akt is cytoplasmic. Sequencing of Akt revealed a high degree of homology to PKA (˜75%) and PKC isozymes (˜50%), a fact which led to its renaming as PKB.
PKB activation involves phosphorylation of two amino acid residues, Ser473 and Thr308. The enzyme is activated by the second messenger PIP3 produced by PI′-3-kinase. PIP3 binds to the pleckstrin homology (PH) domains of PKB, recruits it to the membrane where it is phosphorylated and converted to its activated form. Since PKB activation is PI′-3-kinase dependent, the persistent activation of certain protein tyrosine kinases, such as IGF-1 receptor, EGF receptor, PDGF receptor, pp60c-Src, and the like, leads to the persistent activation of PKB which is indeed encountered in many tumors. Deletions in the gene coding for the tumor suppressor PTEN also induce the persistent activation of PKB/cAkt since it is the negative regulator of this enzyme. Also, PKB is overexpressed in 15% of ovarian cancers, 12% of pancreatic cancers and 3% of breast cancers, and was shown to produce a survival signal that protects cells from apoptosis thus contributing to resistance to chemotherapy.
PKB has emerged as a crucial regulator of widely divergent cellular processes including apoptosis, proliferation, differentiation and metabolism. Disruption of normal PKB/Akt signaling has now been documented as a frequent occurrence in several human cancers and the enzyme appears to play an important role in their progression (Nicholson and Anderson, Cellular Signalling 14, 381, 2002). Therefore PKB is, in principle, an attractive drug target for the treatment of cancer. Ideally, a drug that inhibits PKB should cause both cell cycle arrest and promote appoptosis. Such activity would result in increased cell death of tumor tissue where PKB is elevated, and in decreased resistance to chemoterapy agents.
These molecular properties of PKB and its central role in tumorigenesis, implies that this protein kinase may be an attractive target for novel anti-cancer agents. To date no specific inhibitors of PKB are known in the art, nor are any of the disclosed inhibitors of protein kinases A and C known to act on PKB.
Hidaka H. et al. (Biochemistry, 32, 5036, 1984) describe a class of isoquinolinesulfonamides having inhibitory activity towards cyclic nucleotide dependent protein kinases (PKA and PKG) and protein kinases C (PKC). The same class of compounds is claimed in EP 061673, which discloses said compounds as having cardiovascular activity. Additional derivatives of isoquinolinesulfonyl were disclosed by Hidaka in EP 109023, U.S. Pat. No. 4,456,757, U.S. Pat. No. 4,525,589, and U.S. Pat. No. 4,560,755.
Antitumor activity has been suggested for some of these isoquinolinesulfonamides. Martell R. E. et al. (Biochem. Pharm., 37, 635, 1988) found effects of two isoquinolinesulfonamides, namely 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7) and N-[2-guanidinoethyl]-5-isoquinolinesulfonamide (HA-1004), which have a certain selectivity for PKC and cyclic nucleotide dependent protein kinases, respectively, on calcitriol-induced cell differentiation. Further, Nishikawa M. et al., Life Sci., 39, 1101, 1986), demonstrate that the same compound H-7 inhibits cell differentiation induced by phorbol diester.
International PCT application WO 93/13072 discloses 5-isoquinolinesulfonamide derivatives as protein kinase inhibiting agents wherein the claimed compounds all contain two sulfonyl moieties.
Other classes of compounds known in the prior art (EP-A-397060, DE-A-3914764 and EP-A-384349) showed the capacity of inhibiting protein kinases, however, said compounds have a chemical structure which is totally different from that of the compounds of the present invention. In addition, international PCT application WO 98/53050 discloses short peptides derived from the HJ loop of a serine/threonine kinase which modulate the activity of serine/threonine kinases.
The minimal consensus sequence for efficient phosphorylation by PIM was found by Alessi et al. (Fed. Eur. Biochem Soc., 399, 333, 1996). This is a 7-mer motif faith the most active peptide substrate having the sequence Arg-Pro-Arg-Thr-Ser-Ser-Phe (SEQ ID NO: 1). International application WO 97122360 discloses certain PKB substrate peptides having 7-amino acids length, useful as substrate for measuring PKB activity.
Obata et al. (J. Biol. Chem., 17, 36108, 2000) described the use of an oriented peptide library approach to determine optimal amino acid sequence of the PKB substrate. All the substrates identified contained the known motif having the sequence Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr (SEQ ID NO: 2).
Ricouart et al. (J. Med. Chem. 1991, 34, 73-78), described conjugates of isoquinolinesulfonamides and peptides for the inhibition of PKA. Loog et al. (Bioorganic and Medicinal Chemistry Letters 1999, 9, 1447-1452), described a chimera with adenosine and peptides for the inhibition of PKA and PKC. The inhibition obtained with the disclosed compound is very poor. Schlitzer et al. (Bioorganic and Medicinal Chemistry, 2000, 1991-2006) deal with a small molecule linked to non-peptidic long chain moieties that are supposed to replace the peptide part of the substrate. The disclosed compounds show poor inhibitory activity.
Parang et al. (Nature Structural Biology 8, 37, 2001), describe peptide-ATP bisubstrate analogs of a protein kinase A inhibitor, wherein ATP is linked to a protein kinase peptide substrate. Nevertheless, this approach has a limitation of suboptimal pharmacokinetic properties. WO 01/70770 discloses bisubstrate inhibitors for the insulin receptor tyrosine kinase and a specific potent and selective inhibitor comprising an ATP-gamma-S linked to a peptide substrate analog via a two-carbon spacer.
Numerous disclosures in the background art and in co-pending International Patent Application WO 01/91754 by one of the present inventors relate to specific isoquinoline derivatives, which are PKB inhibitors. The present invention is directed to novel isoquinoline derivatives and more specifically isoquinoline conjugates, and excludes all known compounds previously claimed for their capacity to inhibit PKB.
The present invention overcomes the limitations of known inhibitors by providing ATP surrogates and peptidomimetics with protein target specificity.